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Fig. 6. Failure of cholesterol and 25-HC to inhibit SREBP-2 processing in mammalian cells expressing Insig-2 mutants. On day 0, Scap-deficient
SRD-13A cells were set up in medium C at 3.5 x 105 cells per 60-mm dish. On day 2, each dish of cells was transfected in medium B with 2 µg of pTK-HSV-SREBP-2, 0.1 µg of pCMV-Scap,
and one of the following pCMV–Insig-2–Myc plasmids: wild-type, 0.34 µg; F115A mutant, 0.64 µg; or T136A mutant, 0.34 µg. After
incubation for 16 h at 37°C, the cells were switched to medium D containing 1% HPCD for 1 h, after which the cells were washed
twice with PBS and switched to medium D containing the indicated amounts of cholesterol (complexed to MCD) or 25-HC (in ethanol).
After incubation for 6 h at 37°C, the cells were harvested, fractionated, and subjected to SDS/PAGE and immunoblot analysis
with anti-HSV-IgG (against SREBP-2), IgG-9D5 (against Scap), and IgG-9E10 (against Insig-2). N and P denote the cleaved nuclear
and precursor forms of SREBP-2, respectively.